WO2004041847A1

WO2004041847A1 - Mesoporous glass as nucleant for macromolecule crystallisation

Info

Publication number: WO2004041847A1
Application number: PCT/GB2003/004875
Authority: WO
Inventors: Naomi Chayen; Larry Hench
Original assignee: Imperial College Innovations Limited
Priority date: 2002-11-07
Filing date: 2003-11-07
Publication date: 2004-05-21
Also published as: EP1565484A1; AU2003283546A1; ES2305534T3; US7252713B2; ATE389667T1; DE60319868D1; EP1565484B1; DK1565484T3; GB0225980D0; US20060154042A1; DE60319868T2

Abstract

A method of facilitating the crystallisation of a macromolecule comprising the step of adding a mesoporous glass to a crystallisation sample wherein the mesoporous glass comprises pores having diameters between 4nm and 100nm and has a surface area of at least 50 m2/g. A method of facilitating the crystallisation of a macromolecule comprising the step of adding to a crystallisation sample a mesoporous glass of the composition Si02; CaO-P205-SiO2 or Na20-CaO-P205-SiO2, wherein each of the Ca, P, Si or Na atoms within the compositions may be substituted with a suitable atom chosen from B, Al, Ti, Mg, or K, and, optionally, the composition may also include heavy elements to enhance X-ray diffraction contrast such as Ag, Au, Cr, Co, Sr, Ba, Pt, Ta or other atom with an atomic number over 20.

Description

MESOPOROUS GLASS AS NUCLEANT FOR MACROMOLECULE CRYSTALLISATION

The present invention relates to the use of a novel nucleant in the crystallisation of macromolecules, and methods of crystallising macromolecules, such as proteins using the novel nucleant.

To date, nucleation of crystals has been facilitated mainly by seeding, epitaxy, charged surfaces or mechanical means (Stura, E.A. In Protein crystallization: techniques, strategies, and tips, (ed Bergfors, T.M.) (International University Line, La Jolla; 1999); McPherson, A. & Shlichta, P. Science 239, 385-387 (1988); Sanjoh, A. & Tsukihara, T. J. Cryst. Growth 196, 691-702 (1999); Sanjoh, A., Tsukihara, T. and Gorti, S. J.Crystal Growth 232 618-628 (2001); Visuri, K. et al. Bio/Technology 8, 547-549 (1990)). Nucleation of crystals the necessary first step in the crystallisation process, which influences it decisively. Consequently, the ability to control it is of primary importance in crystallisation experiments. Nucleation presents a free energy barrier which must be overcome in a specific way, different from the supersaturation conditions which subsequently make crystal growth an energetically favourable process (Feher, G. & Kam, Z. Methods Enzymol. 114, 77-112 (1985)). Formation of nuclei in the bulk of a solution is a stochastic process where protein molecules interact until a critical size aggregate is formed. Any environment that favours a higher local concentration of macromolecules provides a potential nucleation point and may lower the energy barrier for nucleation.

Pilot structural genomics projects show the success rate of getting from clone to structure to be about 10%. Production of crystals suitable for X-ray crystallography is found to be the rate-limiting step (e.g. the Human Proteome Structural Genomics pilot project; Brookhaven National Laboratory, The Rockefeller University and Albert Einstein College of Medicine: http://proteome.bnl.gov/progress.html). It is currently crucial to find methods that will help to overcome this stumbling block.

The ultimate aim would be to find a 'universal' nucleant, which would promote crystallisation of a very wide range of proteins under a very wide range of conditions. Previous studies attempting to find nucleants have been undertaken by introducing candidate substances into crystallisation trials in a controlled manner (McPherson and Schlichta Science 239, 385- 387 (1988); Chayen, N.E., Radcliffe, J.W. & Blow, D.M. Protein Sci. 2, 113-118 (1993); Blow, D.M., Chayen, N.E., Lloyd, L.F. & Saridakis, E. Protein Sci. 3, 1638-1643 (1994); Malkin, A.J., Cheung, J and McPherson, A. (1993) J. Crystal Growth 126 544-554 (concerning "Mcpherson Beads" — latex beads)). Some have been useful for individual proteins but none have yet turned out to be of general use. Various other attempts to induce nucleation on irregular or rough surfaces, or surfaces of special composition (poly-L-lysine, plastic) have also proved generally ineffective (Chayen, N.E. & Saridakis, E. J. Cryst. Growth 232, 262-264 (2001).

Sakamoto et al. Nature 408, 449-453 (2000) and Dusastre Nature 408:417 (2000) describe methods of characterising mesoporous materials and report the structures of some mesoporous materials. They are suggested to be useful in various areas of chemistry such as catalysis and molecular filtration but their use in crystal nucleation is not suggested.

We have found that mesoporous glasses with particular characteristics are useful as crystallisation nucleants. The mesoporous glasses are considered to have pores which may entrap macromolecules, for example protein molecules, and encourage them to nucleate and form crystals. A first aspect of the invention provides a method of facilitating the crystallisation of a macromolecule comprising the step of adding a mesoporous glass to a crystallisation sample wherein the mesoporous glass comprises pores having diameters between 4nm and lOOnm and has a surface area of at least 50 m /g.

In particular embodiments, the mesoporous glass comprises pores having diameters between 4 nm and 70 or 80 nm, 4nm and 40 nm or 7 and 40nm. In the embodiment described in Example 2 in which the mesoporous glass has pores having diameters between 7 and 40nm, the modal pore size is 11.2 nm and the mean pore size is 16nm.

Pore size distributions are calculated using the standard technique in the field, ie from gas (for example N₂ or He) adsorption and desorption isotherm data using the BJH method (Barrett EP, Joyney LG, Halenda PP. J of Am. Chem. Soc. 1951; 73: 373-380), as described in Examples 1 and 2. Using such a method, less than 10%, 5% or 2% of the pores (by number) fall outside the specified size range, for example the range of 4nm to 100 nm. The pore size may show a broad Gaussian distribution across the stated range. A range of pore sizes is considered to be provide good nucleation properties; a narrow pore size distribution, for example in which more than 80, 85 or 90% of the pores have a pore size within the range of Xnm and (X+lOnm) or (X+5nm), in which X lies within the specified size range, is considered not to provide such good nucleation properties.

When used herein the term 'mesoporous glass' includes a glass with unordered, irregularly sized pores whose diameters can range from a few nanometers to tens or hundreds of nanometers. The glass may be a fusion of one or more oxides of silicon, boron, and phosphorous, with one or more oxides of sodium, magnesium, calcium or potassium, and optionally sodium or calcium carbonate. Alternatively and preferably the mesoporous glass with unordered, irregularly sized pores whose diameter can range from a few nanometers to tens or hundreds of nanometers can be made by creating a gel from alkoxide precursors of Si0₂, such as tetramethylorthosilicate (TMOS) or tetraethylorthosilicate (TEOS) hydrolysed with H₂O and a catalyst (such as HNO₃ or HF) to make a mesoporous silica (Si0₂) glass or hydrolysed with H₂O and a catalyst in combination with alkoxide precursors of P₂O₅₎ such as triethoxylorthophosphate (TEP, OP(OC₂H₅)₃), or nitrate precursors of CaO, such as Ca (NO₃)₂, or precursors of Na₂O, such as sodium citrate. Examples include SiO₂, CaO-P₂θ₅-SiO₂ or Na₂0-CaO-P₂0₅- Si0₂. The mesopores in the sol-gel-derived glass arise when the three dimensional network is establishing during gelation and are retained following controlled drying, a technique well known to those skilled in the art and described in Hench (1997) Curr Opin Solid State Mater Science 2, 604-610, Li et al (1991) J Appl Biomaterials 2, 231-239, Hench and West (1990) Chem Rev 90, 33-72.

The pores of the mesoporous glass useful in the present invention have diameters of between 4nm and lOOnm (or, in embodiments, other ranges, as noted above), as determined using a standard gas sorption analysis method such as described in Coleman & Hench (2000) A gel-derived mesoporous silica reference material for surface analysis by gas sorption.1: Textural features Ceramics International 26, 171-178. For example, the pore sizes may be calculated from standard gas (for example N₂) desorption data using the BJH method (see Example 2 and [5]). The mesoporous glass may have a heterogeneous population of pore sizes. The material may have open-ended cylindrical pores, as determined from gas (for example N₂) adsorption desorption isotherms, as known to those skilled in the art and as described in Example 2. The hysteresis loop may be of type HI (Sing KSW, Everett DH, Haul RAW. Pure and Appl. Chem. 1985; 57: 603-619), which indicates that the material contains primarily open-ended and interconnected cylindrical pores which are not closed at the surface.

The mesoporous glass of use in the method of the invention may have a surface area of at least 250 m²/g, 200 m²/g, 150 m²/g, 100 m²/g, or at least 50 m²/g. The surface area is preferably less than 300 m²/g. In an embodiment, the surface area is between 150m /g and 100m /g. The surface area of the mesoporous glass is a measure of both the pore size and the number of pores, and can be increased or decreased during the sol-gel process, as will be appreciated by those skilled in the art, and as described in Hench, L.L. (1998) Sol-Gel Silicas, Hayes Publishing Co, New York, and in Iler, R.K. (1979) The Chemistry of Silica, J Wiley & Sons, New York. The surface area of the mesoporous glass can be calculated using a gas (for example N₂) sorption isotherm as described further in Example 2, or any other equivalent surface analysis methods.

Mesoporous glasses that may be of particular use in the method of the invention are described in Example 1 and in Cook et al (2000) Bioceramics, 13, 625 - 628; Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8; Pereira & Hench (1996) J Sol-gel Sci Tech 7, 59-68; Li et al (1992) Chem Proc Adv Mater 56, 627-633; Lenza et al (2002) J Mater Sci, Mat in Med 13, 837-892; Orefice et al (2001) J Biomed Mater Res 55, 460-467.

It will be understood that where the term "macromolecule" is used, we include any molecule over IkDa. Preferably the macromolecule is a biological macromolecule such as a nucleic acid, and more preferably the macromolecule is a polypeptide. Preferably the polypeptide comprises at least 10, 20 or 50 amino acids, more preferably at least 75, 100, 200, 500 or 1000 amino acids. A second aspect of the invention provides a method of facilitating the crystallisation of a macromolecule comprising the step of adding to a crystallisation sample a mesoporous glass of the composition SiO₂, CaO- P₂O₅-SiO₂ or Na₂O-CaO-P2θ₅-Siθ2, wherein each of the Ca, P, Si or Na atoms within the compositions may be substituted with a suitable atom chosen from B, Al, Ti, Mg, or K, and, optionally, the composition may also include heavy elements to enhance X-ray diffraction contrast such as Ag, Au, Cr, Co, Sr, Ba, Pt, Ta or other atom with an atomic number over 20.

In a preferred embodiment of the second aspect of the invention the mesoporous glass is of the composition Siθ₂, CaO-P₂θ₅-Siθ₂ or Na₂0- CaO-P₂0₅-Si0₂.

CaO-P₂0₅-SiO₂ and Na₂O-CaO-P₂O₅-SiO2 are bioactive mesoporous glasses which have been proposed to be useful in regenerating bone (Saravanapavan and Hench (2001) J Biomed Mater Res. 54, 608-18; Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8 Cook et al (2000) Bioceramics, 13, 625 - 628) The Na₂O-CaO-P₂θ₅-Siθ₂ composition is also bioactive and described in detail in Hench & West (1996) Life Chem Prep 13, 187-241. The bioactive bone-bonding response of mesoporous Si0₂ glass has been described by Hench & Walker (1979) in US Patent No 4,171,544.

Example 1 demonstrates the suitability of mesoporous glasses as a nucleant to facilitate the crystallisation of macromolecules

By 'substituted with a suitable atom' we mean that the atom chosen from the selection offered is of a same valency to that of the substituted atom, eg Mg for Ca, K for Na; alternatively atoms of multiple valence, such as P, can be substituted for Si in the glass networks.

It is preferred that the mesoporous glass of use in the second aspect of the invention comprises pores having diameters between 200nm, 150nm or lOOnm and 2,4, 7, 10, or 20nm. Still more preferably, the pore diameters and distribution thereof are as indicated above in relation to the first aspect of the invention. Thus, in embodiments of the second aspect of the invention the mesoporous glass of use in the method comprises pores having diameters of between 4nm and lOOnm or 7nm and 40nm, as measured using standard methods, for example as described in Example 2.

In relation to the first or second aspect of the invention the distribution of pore dimensions of the mesporous glass of use in a method of the invention has a standard deviation of at least 5nm, 1 Onm or 15nm.

Typically, the mesoporous glass which is of use in the second aspect of the invention is distinguished by the non-uniform distribution of the pore sizes within it. Hence, it is preferred if the pores in the mesoporous glass are not uniform in size, and have a high variability in the pore size.

In a further embodiment of the methods of the invention the mesoporous

glass has interconnected pores. For example, the pores may include open-

ended cylindrical pores, as described in Example 2.

When used herein the term 'interconnected pores' includes a mesoporous glass wherein at least some of the pores are interconnected so that the contents of one pore or cavity are accessible to one or more adjacent or connected pores or cavities and intersect as open pores with the surface. Typically, the interconnected nature of the pores is shared by at least 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70% of the pores of the mesoporous glass. Preferably, the mesoporous glass has a HI classification of pores as described by Sing et al (1985) Pure and Appl Chem 57, 603-619, ie the mesoporous glass contains primarily open-ended cylindrical pores that are interconnected.

The porosity of CaO-P₂0₅-Si0₂ and Na₂0-CaO-P₂0₅-SiO₂ can be varied using a foaming technique. For example, nonfoamed CaO-P₂θ₅-Siθ₂ has a surface area of 146.4 m²/g whereas low foamed CaO-P₂O₅-SiO₂ has a surface area of 239.6 m²/g (Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8). Foaming also alters the average pore size and the 3-D nature of the pores and induces very large pores of lOOμm or greater in addition to the mesopores. Therefore, mesoporous glass of the composition CaO-P₂θ₅-Siθ₂ or Na₂θ-CaO-P₂O₅-Siθ₂ may be made using the techniques described in Sepulveda et al (2002) J Biomed Mater Res. 59, 340-8 or as described in Pereira & Hench (1996) J Sol-Gel Sci and Tech 7, 59-68 or Li et al (1992) Chem Proc Adv Mater 56, 627-633, to" generate a porous substance having a surface area suitable for use in the invention, with an average pore size dimension suitable for use in the invention, and a variability in the average pore size dimensions and having interconnected pores suitable for use in the invention.

Materials such as Sephadex™ beads, alumina powders and zeolites have been tried previously in the crystallisation of macromolecules and were found to be ineffective as general-use nucleants (Chayen, N.E. & Saridakis, E. J. Cryst. Growth 232, 262-264 (2001)). Sephadex™ comprises a meshwork produced by cross-linking of the substituent material. This meshwork effectively creates pores, the size of which are determined by the degree or type of cross-linking. It will be appreciated that Sephadex™ and sand are not included in the present definition of "mesoporous glass". A difference between the porous material useful in the present invention and Sephadex™ is that that the pores in each given type of Sephadex bead are of quasi-uniform size. As described above, the mesoporous glass useful in the present invention comprises pores of variable sizes as defined above; the material has an average pore diameter generated by a fairly wide Gaussian- type spread of pore size. Hence the spread of pore dimension in the porous material is wider than that of cross-linked dextrans.

It will be appreciated that a convenient average pore size for a particular application may depend on the size of the macromolecule to be crystallised. For example, larger macromolecular assemblies (ie with a size larger than 5nm or lOnm or 15nm Stokes' radius) such as virus capsids etc. may require bigger pores (ie, pores with an average size larger than lOnm or 20nm or 50nm in any one plane) than the pore size required by smaller macromolecular assemblies, such as those with a Stokes' radius of 5nm or less.

In a preferred embodiment of the methods of the invention, crystallisation of the macromolecule is induced at a lower critical level of super saturation than that obtained where the mesoporous glass is not added to the sample.

In a further or alternative preferred embodiment, the conditions of the crystallisation sample is one which comprises conditions of supersaturation that are favourable to the crystal growth, but are inadequate or insufficient for spontaneous nucleation. Such conditions are a means to maximise the chances of obtaining crystals during initial screening of crystallisation conditions, and provide a means of growing crystals at metastable conditions, at which the slower growth and the lack of excess and secondary nucleation often provide for growth of larger, better diffracting crystals. Determination of which supersaturation level is inadequate or insufficient for spontaneous nucleation is well known in the art of crystallisation. The level of supersaturation can be determined by setting-up screens covering a range of conditions around the conditions that yield crystals/microcrystals spontaneously.

A third aspect of the invention provides for a method of preparing a mesoporous glass as defined in relation to the first or second aspects of the invention for use as a nucleant in crystallisation comprising fracturing said material into pieces of sub-millimetre dimensions.

The mesoporous glass in relation to the third aspect of the invention may be prepared de novo in pieces of sub-millimetre dimensions by formation of the gel network whilst undergoing agitation.

Preferably, the mesoporous glass is converted into pieces or fragments which are no more than 200μm, 150μm or 100 micron in any dimension, more preferably no more than 75 μm or 50 μm, and still more preferably no more than 25μm in any dimension. More preferably the pieces are no more than 10 micron in any dimension. Advantageously, the pieces of mesoporous glass resemble a fine dust. Conversion of a mesoporous glass into fragments may be achieved by using a diamond or SiC cutter.

By "fracture" we mean that the starting material is rendered into smaller fragments or pieces. Fracture may be by any suitable technique, including cutting with a scalpel or by mechanical means (such as using a diamond cutter) or by breaking smaller pieces off the larger one using tweezers. Typically, the mesoporous glass useful in the present invention cannot be broken by hand in small enough pieces. The surface is mechanically fragile, so grinding or crushing under low loads (for example with vibration) is preferred. It will be appreciated that to be useful as a nucleant in crystallisation, the fragments or pieces should be such that the network of pores within the mesoporous glass is exposed. In other words, the pores should be accessible by suitably sized molecules (ie, those which are not bigger than the size of the pores) when contacted by said molecules. Hence, fragments or pieces of the mesoporous glass are not sealed externally in any way by the fracture process.

The fracture method may be manual (as described above), or may be mechanical or one which employs a motorised machine.

A fourth aspect of the invention is a method of determining the structure of a macromolecule comprising the steps of (i) crystallising the macromolecule in the presence of a mesoporous glass as defined in relation to the first or second aspects of the invention, and (ii) analysing the crystal structure of the crystal produced in step (i).

The porous materials which are useful and/or preferred in the fourth aspect of the invention are those as defined in more detail above. Preferably, the porous material is produced by the method of the third aspect of the invention. The material may be combined with the crystallisation sample components in any order. Preferably, the material is added before nucleation or growth of the crystallisation has started.

Nucleation may be detected by any suitable means; either directly, for example by using a microscope, or indirectly, for example by determining the light scatter characteristics as described in Rosenberger et al J. Cryst. Growth 129:1-12 (1993); Saridakis et al Ada Cryst D 58, 1597-1600 (2002). Methods of analysing the crystal structure of a crystal are well known in the art and are described in detail in (Drenth, J. Principles of protein X-ray crystallography. Springer- Verlag, New- York, 1994).

A fifth aspect of the invention is a chamber suitable for crystallising a macromolecule wherein said chamber comprises (at least in part) a mesoporous glass as defined in relation to the first or second aspect of the invention. The chamber may be formed from the mesoporous glass (at least in part; for example in the form of an insert) or the mesoporous glass may form a coating on the chamber (ie on a portion of the chamber not formed from the mesoporous glass). It will be appreciated that the mesoporous glass is in a portion of the chamber that is in contact with the crystalllisation sample, at least at the start of the crystallisation trial.

By 'a chamber suitable for crystallisation' we include a chamber in a crystallisation plate or multi-well plate, or an individual chamber, as will be well known to those skilled in the art. For example, the "cover slip" from which a drop is suspended in "hanging drop" crystallisation (or on which a drop may sit in a "sitting drop" crystallisation) may be formed from or coated with the mesoporous glass.

The chambers can be made or coated with the mesoporous glass by use of a process similar to that described in Roethen et al (2002) Biomaterials 23, 3871-3878. The coating may be a continuous coating or may be a coating of particles of the mesoporous glass.

A further aspect of the invention provides a fibre, mesh or film comprising a mesoporous glass as defined in relation to the first or second aspect of the invention. A further aspect of the invention provides the use of such a fibre, mesh or film as a crystallisation nucleant, for example in a method of the invention. The fibre may be a fibre formed from a different material which is coated with the mesoporous glass (either as a continuous layer or as particles of the mesoporous glass). Crystals grow on the fibre, which may slowly be pulled through the solution and thus enable continuous growth. Thus, the fibre-form nucleant is capable of being used in a continuous process.

Coating may be performed using, for example, a method similar to, but not limited to, that described by Stamboulin et al (2002) Adv Eng Met 105-109 and Roether et al (2002) Biomaterials 23, 3871-3878.

A sixth aspect of the invention provides a use of a mesoporous glass as defined in relation to the first or second aspects of the invention or a chamber as defined in the fifth aspect of the invention in the crystallisation of a macromolecule.

Preferred macromolecules and suitable and preferred mesoporous glasses are described above.

Mesoporous glasses as defined in relation to the first or second aspects of the invention have not previously been contemplated as nucleants in macromolecule crystallisation. Their usefulness in nucleation may be due to the ability of the macromolecules to be crystallised to encounter a pore of a suitable size, be retained by the pores, and as a result be joined by other similar molecules. The retention of the macromolecules in a pore forms a local concentration suitable for nucleation.

A seventh aspect of the invention provides a kit of parts comprising a crystallisation agent and a mesoporous glass as defined in relation to the first or second aspects of the invention or a chamber as defined in the sixth aspect of the invention.

Storage of the mesoporous glass nucleant should preferably be in the presence of a standard indicating dessicant powder.

By "crystallisation agent" we include any one or more of a range of precipitants such as polymers and organic solvents and crystallisation agents such as salts. Specific examples of suitable precipitants include polyethylene glycol 400, polyethylene glycol 4000, mono-sodium dihydrogen phosphate and ammonium sulphate.

It will be appreciated that the kits of the invention are suitable for all crystallisation methods, including the microbatch, vapour diffusion hanging drop, sitting drop, sandwich drop and the free interface diffusion crystallisation methods. It would also be applicable to insert the glass nucleant into gelled drops.

The kits of the invention may further comprise crystallisation plates or slides and/or filters or crystallisation chambers. Where the kit further comprises crystallisation plates, it is preferred if the plates are multi-well plates, which would be particularly suitable for a high throughput screening system.

In a preferred embodiment of this aspect, the kit further comprises oil for layering over the crystallisation sample. Suitable oils include paraffin such as that available from Hampton Research, CA 92677-3913 USA, catalogue number HR3-411. In an alternative embodiment, the kit may be one which is suitable for screening for the crystallisation of macromolecules. In this embodiment, the kit may further include any one or more of a range of buffers (which may cover a range of pH values) and/or any one or more of a range of salts. Suitable buffers and salts are known in the art of crystallisation and include Na-HEPES pH 7.5, Tris hydrochloride pH 8.5 as buffers, and 0.2M ammonium sulphate and 0.2M sodium acetate trihydrate as salts. Advantageously, the kit may further comprise multi-well crystallisation plates and filters.

An eighth aspect of the invention provides an automated method of crystallising a macromolecule comprising adding a mesoporous glass as defined in relation to the first or second aspects of the invention to a crystallisation trial using an automated dispensing system.

Suitable and preferred mesoporous glasses are as described above.

Advantageously, the crystallisation is part of a screen or optimisation for crystallisation conditions.

The mesoporous glass may be prepared for administration to the trial by any suitable means. Preferably, the mesoporous glass is in the form of fine fragments, of no more than 10 micron in any dimension. In a preferred embodiment, the mesoporous glass is prepared according to the third aspect of the invention.

It will be appreciated that the mesoporous glass may be added as a single grain or particle or piece, or it may be added as a suspension in a liquid.

Ensuring that only a single particle which may be only 10 micron in size is dispensed into an automated trial may be awkward, or may involve an unacceptably high level of error, such that no particle, or too many particles are added. Clearly then, an advantage of the method where the mesoporous glass is added as a suspension is the ability to manipulate the mesoporous glass more easily, especially automatically using an automated liquid dispensing system. Forming a suspension of the mesoporous glass particles in a liquid such that dispensing a particular volume of the liquid is likely to include at least 1 piece or fragment of the mesoporous glass, but not too many pieces or fragments, would facilitate the step of adding the fragment to a crystallisation sample.

Hence, in a preferred embodiment of this aspect of the invention, the mesoporous glass is dispensed by an automated liquid dispensing system as a suspension.

Automated liquid dispensing systems are known in the art of protein crystallisation, and an example is the IMP AX system produced by Douglas Instruments, Hungerford, UK. In this system, several components of the crystallisation sample can be dispensed from separate reservoirs through the same tip into a single sample drop. Suitably, the automated system useful in this aspect of the invention is programmable such that defined volumes and concentrations of liquid or suspension may be dispensed into the crystallisation drop.

The crystallisation trial may be any suitable method, including microbatch (Chayen et al (1990) Appl. Cryst. 23:297; Chayen et al (1992) J. Crystal Growth 122:176) and vapour diffusion. The microbatch method may be modified as described in D'Arcy et al (1996) J. Crystal Growth 168:175- 180 to use a mixture of silicon and paraffin oil over the crystallisation sample. To maintain the level of supersaturation which is suited to crystal growth, and which does not promote nucleation, it is preferred if the oil used is one which does not permit detectable diffusion of water molecules to the oil-air interface, and therefore one which does not promote a concentration of the components of the crystallisation sample. Hence it is preferred if the oil is a branched paraffin in the C20+ range, and not a silicon fluid (such as a polymer of dimethylsiloxane units).

An ninth aspect of the invention provides a crystal obtainable or obtained by the method of the first, second or eighth aspects of the invention.

As described above, it is likely that use of the mesoporous glass in the crystallisation material causes the macromolecule to be crystallised to nucleate within the pore network of the material, and not on the surface of the mesoporous glass. Consequently, the resulting crystal may include the mesoporous glass within the actual crystal structure of the crystal produced. Such crystals may be distinct from crystals obtained by alternative means (such as using a mineral surface to promote nucleation), since the nucleant may form an integral part of the crystal, and not a peripheral part.

A further aspect of the invention provides a use for an automated liquid dispensing system in the method of the eighth aspect of the invention, wherein the mesoporous glass to be dispensed is in the form of a suspension.

A "automated liquid dispensing system" includes any suitable automated dispensing system capable of dispensing a volume of a suspension of the porous material which is between 2 nl and 1 ml, for example between 2 nl and 50 or 20 nl, for example 5 nl or lOnl (for low volume/high throughput trials) or between 0.05 or 0.1 μl and 1ml (for larger volume trials). Preferably the system is one as described above. The fibre, mesh or film of the invention may also be used in an automated system.

The invention will now be described in more detail with the aid of the following, non-limiting Figures and Examples.

Documents referred to herein are hereby incorporated by reference.

Figure 1 : Crystals of alpha crustacyanin (from lobster shell) grown on bioactive CaO-P₂O₅-SiO₂ gel-glass particles. The crystals measure approx 140x80x40 μm.

Figure 2: N₂ sorption isotherms for 58S (mesoporous) and 45S5 (control) glass powders.

Figure 3: Pore size distributions of 58S and 45S5 glasses.

Figure 4: Schematic diagram of a cross-section of a mesoporous glass (non- nucleant) that has a narrow pore size distribution.

Figure 5: Schematic diagram of a mesoporous glass nucleant with a broad distributino of pore diameters, from 7 to 40 nm.

Example 1: A method of crystallising crustacyanin using CaO-P₂O₅- SiO₂ mesoporous glass as a nucleant.

The materials tested as nucleants were bioactive CaO-P2O5-SiO2 gel-glass particles. One (58S) has a distribution of pores as described in Example 2 and the other does not have detectable pores (45S5; control), as described in Example 2.

The CaO-P₂0₅-Si0₂ (58S) mesoporous glass nucleant was prepared by hydrolysing teraethylorthosilicate (TEOS) with H₂O in the presence of HNO₃ as a catalyst. The precursor of the P₂O₅ was triethoxylorthophosphate (TEP) and the CaO precursor was Ca(N0₃)₂.4H₂O. The gel was dried at 60°C and 130°C and stabilised at 100°C to remove water from the pores and pore surfaces.

The bioactive 45S5 glass was prepared by melting SiO₂, P₂O₅, CaCO₃ and Na₂C0₃ in a Platinum crucible at 1350°C and quenching the molten glass into water to form a frit follwed by crushing to form the non-porous particles.

The protein used (alpha crustalcyanin) tends to produce showers of crystals overnight which are disordered. It was tested whether, if using the glass nucleants, large single crystals would be obtained under conditions of lower supersaturation, which would otherwise produce clear drops.

Two sets of Control drops were set up:

(1) At supersaturated conditions which give the showers of crystals.

(2) At lower supersaturation conditions (i.e. lower precipitant concentration or what is termed as metastable conditions).

Other drops from the same protein preparation were set up at the same conditions as (2) but one set of drops contained the CaO-P₂O₅-SiO₂ mesoporous glass (1 or 2 grains added to the drop), while another set of drops had non porous glass grains added to the drops. Control (1) gave showers of crystals overnight as expected. Control (2) remained clear indefinitely

The drops containing the porous nucleant produced large single crystals after 2-3 days. Figure 1.

The drops containing the (non porous) 45S5 bioactive glass remained clear as if no nucleant had been added.

The methods used were vapour diffusion and microbatch with in each case 2 μl drops. Trials were incubated at 10 and 4 °C. Supersaturated conditions which gave rise to crystals overnight were:

20mg/ml protein 15% PEG monomethyl ether 0.1M MES buffer pH 6.5 and lOmM Zinc chloride.

Metastable conditions into which the nucleants were inserted were: 20mg/ml protein 12.5% PEG monomethyl ether 0.1M MES buffer pH 6.5 and lOmM Zinc chloride.

Glass characterisation methods

N₂ adsorption

The gas adsorption was performed on a six-port Quantachrome AS6B-KR Autosorb gas sorption system using N₂ gas. The instrument determined isotherms volumetrically by a discontinuous static method at 77. K (Manual for Quantachrome N₂ Adsorption. Autosorb 6®, Quantachrome Corp. USA, 1997). N₂ with cross-sectional area of 0.162nm² was used as the adsorptive gas (Lowell and Shields Powder Surface Area and Porosity 1984). Further details are given in Example 2.

Example 2: Measurement of pore characteristics

N₂ adsorption The gas adsorption was performed on a six-port Quantachrome AS6B-KR Autosorb gas sorption system using N₂ gas. The instrument determined isotherms volumetrically by a discontinuous static method at 77.4K [1]. N₂ with molecule cross-sectional area of 0.162 nm was used as the adsorptive gas [2].

Prior to N₂ sorption, all samples were degassed (i.e. exposing the monoliths to a vacuum pressure lower than lPa at an elevated temperature) at 100° C overnight (approx. 16- 20 hours). This is done to remove physically adsorbed gases from the sample surfaces, in particular water vapour [2]. The specific surface areas were estimated in relation to the masses of the outgassed samples.

Each isotherm comprised a minimum of 20 adsorption and 20 desorption points measured at equilibrium. At least 4 adsorption points in the relative pressure range 0.05<P/P₀<0.30 (where P₀ is the saturated vapour pressure) were used in the calculation of the BET surface area [3] [4]. In each case, it was ensured that the slope and the intercept of the BET plots were positive and that the linear regression correlation coefficients were not less than 0.99991 [1]. Three isotherms were collected for each composition to ensure that the data was representative. Estimates of specific pore volume were obtained from the amount of nitrogen taken up by the sample in the relative pressure range 0.994 < P/P₀ < 0.999 [1]. Pore size distributions were calculated from the desorption data using the BJH method [5].

Results - N₂ adsorption Isotherms

The isotherms shown in Figure 2 are those obtained for 45 S5 melt-derived glass and 58S sol- gel glass powders. The melt-derived glass displayed a very low adsorption, mainly monolayer formation followed by saturation, revealing negligible levels of porosity. In contrast, the isotherm for 58S sample corresponds to Type IV of the IUPAC classification indicating that the porous adsorbents possess pores in the radius range of 20 to 500 A (diameter range of 4 to 100 nm) (i.e. the samples are mesoporous) [3]. Hysteresis in the multi layer region of the isotherm is associated with capillary condensation of N₂ during filling and emptying of mesopores in the structure. The hysteresis loop seen here is of type HI [3]. This indicates that the materials tested contain open-ended cylindrical pores.

The pore size distribution for the gel-glasses, calculated using the BJH model [5] on the desorption branch of the N₂ isotherm is depicted in Figure 3.

Summary

References

[1]. Manual for Quantachrome N₂ Adsorption. Autosorb 6®, Quantachrome Corp. USA, 1997.

[2]. Lowell S, Shields JE. Powder Surface Area and Porosity. Chapman and Hall 1984. [3]. Sing KSW, Everett DH, Haul RAW. Pure and Appl. Chem. 1985; 57: 603-619.

[4].Brunauer S, Deming LS, Deming WE, Teller E. J ofAm. Chem. Soc. 1940; 62: 1723-1732.

[5].Barrett EP, Joyney LG, Halenda PP. JofAm. Chem. Soc. 1951; 73: 373-

380

Claims

1. A method of facilitating the crystallisation of a macromolecule comprising the step of adding a mesoporous glass to a crystallisation sample wherein the mesoporous glass comprises pores having diameters between 4nm and lOOnm and has a surface area of at least 50 m²/g.

2. A method of facilitating the crystallisation of a macromolecule comprising the step of adding to a crystallisation sample a mesoporous glass of the composition SiO₂; CaO-P₂O₅-SiO₂ or Na₂0-CaO-P₂O₅-SiO₂, wherein each of the Ca, P, Si or Na atoms within the compositions may be substituted with a suitable atom chosen from B, Al, Ti, Mg, or K, and, optionally, the composition may also include heavy elements to enhance X-ray diffraction contrast such as Ag, Au, Cr, Co, Sr, Ba, Pt, Ta or other atom with an atomic number over 20.

3. A method according to Claim 2 wherein mesoporous glass is of the composition SiO₂; CaO-P₂O₅-SiO₂ or Na₂O-CaO-P₂θ₅-Siθ₂.

4. A method according to Claims 2 or 3 wherein the mesoporous glass comprises pores having diameters between 2nm and 200nm.

5. A method according to Claim 4 wherein the diameter of the pores has a standard deviation of at least lOnm.

6. A method according to any of the above claims wherein the mesoporous glass has interconnected pores that intersect with the surface of the glass.

7. A method according to any one of the previous claims wherein crystallisation of the macromolecule is induced at a lower critical level of super saturation than that obtained where the mesoporous glass is not added to the sample.

8. A method of preparing a mesoporous glass as defined in relation to claim 1 or 2 for use as a nucleant in crystallisation comprising fracturing said material into pieces of sub-millimetre dimensions.

9. A method according to claim 8 wherein the pieces are no more than 200 micron in any dimension.

10. A method according to claim 9 wherein the pieces are no more than 100 micron in any dimension.

11. A method according to any one of claims 8 to 10 wherein the fracture is by cutting with a scalpel or mechanical means (diamond cutter) or breaking smaller pieces off a larger one using tweezers, or crushing with a low load.

12. A method of determining the structure of a macromolecule comprising the steps of:

(i) crystallising the macromolecule in the presence of a mesoporous glass as defined in relation to claim 1 or 2; and,

(ii) analysing the crystal structure of the crystal produced in step (i).

13 A chamber suitable for crystallising a macromolecule, or a fibre, film or mesh, wherein said chamber, fibre, film or mesh comprises a mesoporous glass as defined in relation to claims 1 or 2

14 A chamber, fibre, film or mesh according to claim 13 wherein the mesoporous glass forms a coating on the chamber, fibre, film or mesh.

15. Use of a mesoporous glass as defined in relation to claim 1 or 2 or a chamber, fibre, film or mesh according to claims 13 or 14 in the crystallisation of a macromolecule.

16. A kit of parts comprising a crystallisation agent and a mesoporous glass as defined in relation to claim 1 or 2 or a chamber as defined in claim

13 or 14.

17. An automated method of crystallising a macromolecule comprising adding a mesoporous glass as defined in relation to claim 1 or 2 to a crystallisation trial using an automated dispensing system.

18. A method according to Claim 17 wherein the crystallisation is in a screen or optimisation.

19. A method according to Claim 17 or 18 wherein the mesoporous glass is added as a suspension in a liquid.

20. A method according to Claims 1 to 3 or 12 or 17 to 19 or a use according to Claim 13 or a kit according to any one of Claims 14 to 16 wherein the mesoporous glass as defined in relation to claim 1 or 2 is prepared according to the method of any one of Claims 8 to 11.

21. A crystal obtainable or obtained by the method of any one of Claims 1 to 3 or 17 to 21.

22. A method according to Claim 1 to 3 or 12 or 17 to 19 or a use according to Claim 13 or a crystal according to Claim 21 wherein the macromolecule is a biological macromolecule.

23. A method or use according to Claim 22 wherein the macromolecule is a protein.

24. Use of an automated liquid dispensing system to dispense a porous mesoporous glass according to the method of Claim 19.